Abstract ! The objective of this project is to advance the development of a novel approach to healing chronic diabetic foot ulcers (DFUs), which are a major and growing health concern for which existing treatments are often ineffective. Normal wound healing involves a coordinated cascade of events that are stimulated in part by the hypoxia that results from injury to the vasculature at the wound site. These events include angiogenesis, macrophage recruitment, inhibition of apoptosis, and the expansion and mobilization of fibroblasts and keratinocytes for re-epithelialization. In chronic wounds, the normal response to hypoxia is impaired, and many of these cellular processes are hindered. Our approach involves the therapeutic modulation of a protein and a microRNA (miRNA) that are involved in these hypoxia-induced pathways by the use of oligonucleotides acting by two different mechanisms, RNA interference (RNAi) and antisense. RNAi is achieved by use of SomaGenics? proprietary sshRNA (synthetic short hairpin RNA) design, which eliminates sense-strand off- target effects and has been shown to be highly effective in treating chimeric mice infected with hepatitis C virus. In previous SBIR-supported work, we identified oligonucleotides that potently inhibit the protein and miRNA targets in vitro and demonstrated that when these RNAs are incorporated into a medicated bandage by a layer-by-layer (LbL) process and applied to full-thickness wounds of diabetic mice, healing is significantly accelerated and regrowth of vasculature is enhanced. Treatment was also shown to increase levels of downstream factors promoting angiogenesis and to increase the mobility of keratinocytes. The lack of a perfect animal model for DFU healing has led to a consensus among researchers that efficacy and safety should be demonstrated in two diabetic animal models before moving to human trials. Therefore, in this new Phase I proposal, we will validate our mouse results in the neuroischemic diabetic rabbit ear model in collaboration with Dr. Aristidis Veves (Beth Israel Deaconess Medical Center and Harvard Medical School), a leading expert in the rabbit ear model and authority on DFU healing in general. We will continue to employ the bio-erodible LbL drug delivery platform that we have adopted in collaboration with Dr. Paula Hammond (MIT), who has developed this platform. If this Phase I is successful as we expect, in Phase II we plan to perform extensive lead optimization and refinement of our LbL delivery method, including further development of chemical modification patterns that stabilize the RNAs against ribonucleases present in the wound bed without compromising potency or introducing undesired immunostimulatory activity. Optimization will also include defining optimal proportions of the two oligonucleotides in the LbL formulation. We will develop a scalable manufacturing process for large-scale production of LbL-formulated bandage material. Finally, preliminary but carefully designed safety/toxicity studies on a third animal model will be performed. !